Structural and Functional Studies of Designed Antimicrobial & Anti-Inflammatory Peptides:

Abstract

Multi-drug resistant (MDR) bacteria or superbugs have become a global threat to human life due to their inherent resistance to antibiotics. In this context, antimicrobial peptides (AMPs) have emerged as promising alternatives owing to their unique structural and functional characteristics. In fact, AMPs are less susceptible to bacterial resistance than traditional antibiotics. Structural insights into these AMPs using NMR spectroscopy have shown a large correlation between the structure and biological properties of different classes of AMPs, a fact utilised for designing newer peptide-based antibiotics. Approaches aiming to improve the potency and stability of peptides necessitate understanding the sequence-structure-function relationship in developing and designing AMP-based drugs. In contrast to the significant progress over the last decade, particularly regarding AMP optimization, delivery aspects of AMPs have been less addressed, despite the potential of delivery systems to improve AMP performance. This works aims to (i) Characterize, study, and overcome the limitations associated with peptide-based therapy using the technique of de novo antimicrobial peptide designing, which are effective against plant and animal disease-causing pathogens. The structural insights of AMPs interacting with bacterial membrane components will also pave the way to develop novel antimicrobial biopeptides. Initial investigation of the LK series of peptides led to the designing of short AMPs that would economize the cost and time of synthesis associated with the largescale synthesis of AMPs to be used as therapeutic molecules. Of the five designed peptides, P2-P6, P4, and P5 were effective against bacterial strains belonging to the ESKAPE group as well as human fungal opportunistic pathogens. Structural correlation revealed that secondary structures are not a prerequisite for functional attributes in the case of short peptides. Additionally, the emphasis was made to understand the phenomenon of peptide synergism by studying the enhanced potency of VG16KRKP and KYE28, 16- and 28-mer de novo designed peptides, respectively, when used in conjunction with one other. NMR spectroscopy of the complex formed between these two peptides and their mutated analogs led to discovering an unusual peptide complex, characterized by the formation of a bulky hydrophobic hub, stabilized by aromatic zippers highlighting this structure as key for elevating antimicrobial potency of the peptide combination. Furthermore, insights into the activity of the anti-inflammatory peptide KYE28 and its PEGylated variants have been investigated to correlate their ability to disintegrate LPS aggregates and the ability of PEGylation for reducing toxicity and proteolytic susceptibility of AMPs. (ii) Additionally, it also involves probing and studying potential nano-materials as AMP delivery systems such that AMPs can be used for treating systemic diseases. In this context, it is worth mentioning that AMPs need to penetrate the cell to target the microbes. Various techniques have been developed for modifying natural AMPs, making them better suited to cross the cell membrane and get explicitly delivered to the target bacterial membrane. Gold nanoparticle conjugation and poly (ethylene glycol) conjugation are two such delivery techniques studied in this work. NMR-based structural insights depicted that both the conjugation techniques increase the peptides' bioavailability without hampering their functional attributes.